My long-term goal is to understand the molecular basis underlying the fundamental differences between rod and cone phototransduction and to apply the knowledge to other G-protein signaling systems. Rod and cone photoreceptors use similar schemes to convert light into neuronal signals. Rods are more sensitive but respond slower than cones. The phototransduction pathways involve different but related genes. The first two aims of this application exploit such differences in rod and cone gene expression to highlight the rate-limiting step in rod recovery. The third aim focuses on the characterization of GRK7, a novel member of the G-protein coupled receptor kinase (GRK) family and a potential cone opsin kinase in human. Aim 1 is to over-express the RGS9-1/GBeta5-L GAP complex in mouse photoreceptor using a 4.4 Kb mouse opsin promoter by generating RGS9-l and GBeta5-L transgenic mice respectively and by crossbreeding them. The effects of over-expression on rod phototransduction will be examined by measuring the rate of transducin GTP hydrolysis and by single rod suction recordings under dim flash conditions. "Paired-flash" analyses of cone-derived electroretinography (ERG) will be used to assess the effect of RGS9-1/GBeta5-L over-expression on cone phototransduction. Aim 2 is to over-express farnesylated (normal) GRK 1 and geranylgeranylated (mutant) GRK 1 in mouse photoreceptors using transgenesis as described above. The effects of over-expression will be tested by measuring the rate and the sites of rhodopsin phosphorylation using conventional biochemical techniques and advanced mass spectrometry. Single rod recordings and "Pair-flash" cone derived ERG analyses will be used to examine the physiological effects on rods and cones, respectively. Aim 3 is to express and compare the activities of purified recombinant GRK1 and GRK7 on activated rhodopsin and to generate transgenic mice ectopically expressing human GRK7 in mouse photoreceptors on a GRK1 null background. Characterizations of hGRK7 transgenic mice will be performed as described for aim 2. Results obtained from the proposed in vitro and in vivo experiments determine whether or not GRK7 substitutes for GRK1, a corollary for the normal photopic vision reported by human oguchi disease patients with defective GRK1. Yeast two-hybrid screen using GRK7 as a bait on a retinal cDNA library will be performed to identify potential GRK7 substrates and/or regulators in the retina. These approaches shall generate useful reagents in the field of signal transduction, significantly advance our knowledge on the recovery of rod and cone phototransduction and provide valuable insight into the physiological functions of GRK7.